Process for increasing glycine levels in the brain and spinal cord

ABSTRACT

The level of glycine in the brain and in the spinal cord is regulated by administering a neutral amino acid composition to a human wherein increased or decreased brain and spinal cord levels of glycine is effected when the composition contains increased or decreased amounts of threonine or a precursor of L-threonine. The neutral amino acid composition can be administered alone or concomitantly with a drug which either has the undesirable side effect of suppressing glycine-mediated neurotransmission or whose therapeutic efficacy is enhanced by increasing glycinergic neurotransmission.

The Government has rights in this invention pursuant to Grant NumberAM-14228 awarded by the National Institute of Health.

This is a division, of application Ser. No. 036,925 filed May 7, 1979,now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to a method and composition for regulating(increasing and decreasing) the level of glycine in the brain and in thespinal cord.

Prior to the present invention, it was thought that glycine was formedin vivo, in the mammalian central nervous system, from serine by theremoval of a hydroxyl group. It had also been proposed that glycinecould be produced in vivo by the aldol cleavage of threonine to yieldglycine and acetaldehyde, Meltzer et al, J. Biol. Chem., 107, pp 461-474(1952). In order to test this hypothesis, experiments were conductedwith chicks that were fed a diet containing L-threonine to determine theeffect of L-threonine on glycine formation in vivo. As reported by D'Melo at Nutr. Metabol., 15, pp 357-363 (1973), L-threonine was found tohave no appreciable influence on plasma glycine levels. Accordingly, itwas concluded that threonine is not readily degraded to glycine and thatit cannot act as a precursor of glycine.

Glycine is known to be an inhibitory neurotransmitter in the brain and,especially, the spinal cord. Prior to the present invention, there areno drugs or other treatments for increasing the amount of glycinepresent in synapses. It is known that conditions or treatments thatdecrease neuronal glycine levels such as spinal cord lesions or thatblock glycine's interactions with its receptors such as the poisonstrychnine cause spasticity and hyper-reflexia. Brain glycine levelsalso are depressed when animals receive anti-psychotic drugs chronicallyand glycine-mediated neurotransmission may be involved in the actions ofsome tranquillizing drugs (like the benzodiazopines).

It would be highly desirable to provide a means for increasing ordecreasing the amounts of glycine in the brain and in the spinal cord.Furthermore, it would be desirable to provide such a means which isbiochemically specific and which lacks the undesirable side effects ofanti-spasticity drugs (such as mephenesin analogs, that cause jaundice,nystagmus, and nausea; or dantrolene, which produces weakness). Such ameans would be useful in conditions associated with spasticity such asbackache or muscle strain. In addition, such means could be utilized inconjunction with anti-psychotic drugs that have the undesirable sideeffect of lowering brain glycine levels, or with tranquillizing drugsthat act as glycine receptors.

SUMMARY OF THE INVENTION

The present invention provide a method and composition for treatingdiseases associated with deficiency of glycine in the brain or spinalcord. This invention is based upon the discovery that the administrationof L-threonine, an essential dietary amino acid, causes increasedglycine levels in the brain and the spinal cord. The L-threonine can beadministered alone or in admixture with other amino acids; as the acid,its salts, or as a small peptide; with or without drugs, in order toraise brain and spinal cord glycine levels, and, thereby, to treatdiseases associated with deficiency of glycine in the brain or spinalcord. By varying the proportion of tryptophan, another amino acid, inthe mixture, the synthesis and synaptic release of serotonin, anotherbrain neurotransmitter, can similarly be controlled. In addition, byvarying the proportion of tyrosine, another amino acid, the release ofdopamine and/or norepinephrine into neuronal synapses can be controlled.Increased intrasynaptic dopamine levels are obtained after tryosineadministration only when the dopamine-releasing neurons are active,i.e., are firing frequently. Increased synaptic norepinephrine levelsare obtained by giving tyrosine regardless of whether thenorepinephrine-releasing neurons are or are not especially active.Decreases in dopamine and norepinephrine release into synapses can beobtained by lowering brain tyrosine levels by administering neutralamino acid compositions low in tyrosine levels. Decreases in serotoninrelease can similarly be obtained by lowering brain tryptophan levels.Phenylalanine can, in low doses, be used in place of tyrosine. Glycinesynthesis can also be decreased by administering meutral amino acidmixtures that lack threonine and which thus reduce threonine levels inthe central nervous system.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

In accordance with this invention, L-threonine is administered to apatient either alone or in combination with one or more drugs thereby toincrease the levels of glycine in the brain and spinal cord. Serotoninrelease also can be controlled at the same time by varying theproportion of tryptophan present in the amino acid mixture. Dopamineand/or norepinephrine release into synapses also can be controlled byvarying the proportion of tyrosine and/or its precursor phenylalanine inthe L-threonine-containing composition. In order to increase dopaminerelease, it is necessary that the dopamine-releasing neurons in thepatient's brain be relatively active, i.e., are firing frequently, suchas is the case in patients with Parkinson's Disease. However, release ofnorepinephrine or serotonin into synapses can be varied using amino acidmixtures whether or not the norepinephrine-releasing orserotonin-releasing neurons are especially active. Glycine synthesis andrelease can be diminished by administering neutral amino acids otherthan threonine, since such compounds reduce brain and spinal cordthreonine levels by competing with it for uptake at the bloodbrainbarrier.

The composition of the amino acid mixture that is utilized depends uponthe nature of the illness in the patient that is to be treated. Whenthere is need to increase glycine release without increasing that ofserotonin, dopamine or norepinephrine, L-threonine is administered, notincluding serotonin's precursor, tryptophan or the precursors ofdopamine or norepinephrine, in doses ranging between 5 mg/kg and 200mg/kg body weight. This therapy is useful, alone or as an adjunct todrug therapies, in treating muscle spasticity, (e.g., in paraplegia,multiple sclorosis, or vertabrae disc abnormalities), certain types oftremor, muscle cramps, or certain conditions requiring a tranquilizer.When there is a need to increase dopamine and/or norepinephrine releasedinto neuronal synapses, tyrosine is included in the L-threoninecomposition in amounts generally between about 2.5 and 100 mg/kg bodyweight. In some situations, phenylalanine can be used as a substitutefor tyrosine, inasmuch as much of this amino acid is converted totyrosine in the liver, and released into the blood stream for uptakeinto the brain. However, plasma phenylalanine levels should be less thanabout double those of tyrosine, since at the higher levels,phenylalanine competes with tyrosine for uptake into the brain, and caninhibit the enzyme tyrosine hydroxylase.

When there is need to sustain or increase brain serotonin levels whileincreasing glycine release, these compositions also contain tryptophan.This combination is especially useful in treating certain types ofdepression, or sleep disorders. Generally, the tryptophan is utilized inthese compositions in amount of between about 2.5 and 100 mg/kg bodyweight.

When there is need to decrease glycine release (by decreasing theavailablity of threonine in the brain or spinal cord), such otherneutral amino acids as leucine, isoleucine, valine, tyrosine,phenylalanine, or tryptophan are given, in combined amounts of 10 mg/kgto 50 mg/kg, without threonine. This treatment is useful in conditionsassociated with muscular flaccidity.

The L-threonine, tyrosine, phenylalanine or tryptophan, or other aminoacids can be administered as free amino acids, esters, salts, natural orsynthetic polymers or as constituents of foods. The route ofadministration can be oral or parenteral, e.g., intravenous. TheL-threonine can be administered with drugs in order to increase glycinelevels in the brain and spinal cord during drug treatment. The drugsutilized in the compositions of this invention with L-threonine can havethe effect of either lowering or raising glycine levels in the brain orspinal column. In the first instance, the L-threonine corrects theglycine deficiency caused by the drug. In the latter instance, theL-threonine increases the effectiveness of the drug's action ofenhancing glycinergic neurotransmission so that it is thereby possibleto reduce the dosage of the drug. For example, L-threonine can beadministered with haloperidol or thorazine (which lower glycine levels)to treat psychosis, with mephenesin analogs or dantrolene to treatmuscle spasticity, with benzodiazepines to attain tranquillization, etc.

The following examples illustrate the present invention and are notintended to limit the same.

EXAMPLE I

This example illustrates that levels of glycine in the brain and spinalcord can be increased by administering L-threonine to an animal.

Male Sprague-Dawley rats (Charles River Breeding Laboratories,Wilmington, MA) weighing 150-175 g were given ad libitum access to tapwater and a formula diet and Charles River rat maintenance maintainedunder light for twelve hours per day. The rats were given L-threoninedissolved in saline intraperitoneally at the levels set forth in TableI. One hour after administration, the rats were killed. The brains andspinal cords were removed, frozen on dry ice and subsequentlyhomogenized. The glycine (GLY) and threonine (THR) contents of thehomogenates were assayed using a Beckmann amino acid autoanalyzer.

L-threonine (Grand Island Biological Co., Long Island, N.Y.) which ispoorly soluble in water was dissolved in dilute NaOH; the solution wasthen buffered to pH 7.4 with hydrochloric acid and brought to a knownvolume with saline. This yielded a fine suspension that was suitable forinjection.

The results are set forth in Tables I and II.

                  TABLE I                                                         ______________________________________                                        Brains                                                                        Dose of                                                                       Threonine      THR      GLY                                                   (mg/kg)        (micromoles per gram ± SEM)                                 ______________________________________                                        Control        .27 ± .02                                                                           .50 ± .09                                          50             .41 ± .09                                                                           .55 ± .11                                          100            .47 ± .02                                                                           .63 ± .05                                          200            .54 ± .05                                                                           .64 ± .08                                          400            .65 ± .02                                                                           .62 ± .04                                          ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        Spinal Cords                                                                  Dose of                                                                       Threonine      THR      GLY                                                   (mg/kg)        (micromoles per gram + SEM)                                    ______________________________________                                        Control        2.6 ± .04                                                                           .49 ± .04                                          50             2.6 ± .11                                                                           .56 ± .04                                          100            3.1 ± .16                                                                           .67 ± .04                                          200            3.1 ± .07                                                                           .80 ± .04                                          400            3.3 ± .10                                                                           1.09 ± .04                                         ______________________________________                                    

As shown in Tables I and II brain and spinal cord levels of glycine, asa result of L-threonine administration, a much greater effect is seen inspinal cords than in brains. This is compatible with the knowndistribution of glycine-releasing neurons.

I claim:
 1. The process for increasing the amount of glycine in thebrain and spinal cord of a patient undergoing haloperidol therapy andsuffering from a natural or unduced deficiency of glycine in the brainor spinal cord which comprises administering to said patient aneffective amount of a neutral amino acid composition comprisingL-threonine, a prcursor of L-threine or mixtures thereof.
 2. Acomposition of matter in unit dosage form comprising haloperidol and aneutral amino acid composition containing L-threonine in an amountsufficient to effect an increase in glycine levels in the brain orspinal cord when administered to a patient.
 3. The composition of claim2 which includes tyrosine, phenylalanine or mixtures thereof in anamount sufficient to increase dopamine and/or norepinephrine releasedinto synapses.
 4. The composition of claim 2 which includes tryptophanin an amount sufficient to increase brain serotonin levels.